1. Field of the Invention
The present invention relates to isoforms of the family of transcription factors known as Stat (signal transducers and activators of transcription). In particular, the present invention provides attenuated and dominant negative variants of human Stat6, which are designated Stat6b and Stat6c and which have differential effects on the modulation of Stat6 activity in cells.
2. Background Art
The Stats have been evolutionarily conserved from Drosophila to humans. Physiologically, Stat signaling pathways have been correlated with pleiotropic and mitogenic functional responses induced by a variety of growth factors, cytokines and interferons. In particular, Stat6 activation correlates with functional responses induced by interleukin-4 (IL-4) (22), interleukin-13 (IL-13) (25) and platelet-derived growth factor (PDGF) (26).
IL-4 is a pleiotropic cytokine that plays a prominent role in the regulation of inflammatory and cell-mediated immune responses (1). IL-4 treatment induces tyrosine phosphorylation of the IL-4 receptor, designated IL-4Rxcex1 (11, 12), a member of the hematopoietin receptor superfamily (13, 14). Unlike several members of the hematopoietin receptor superfamily, IL-4Rxcex1 is ubiquitously expressed on cells of hematopoietic and nonhematopoietic origin. IL-4Rxcex1 activation results in tyrosine phosphorylation of multiple substrates including Jak1, Jak3 (15, 16), IRS-1 (17), IRS-2/4PS (18) and Stat6 (13, 14, 19, 20). Phosphorylation of specific tyrosine residues within the two GYKXF motifs present in the IL-4Rxcex1 has been proposed to be crucial for binding to and activation of Stat6 (13, 22).
Selective activation of the Stats results in dimerization and translocation to the nucleus, where each interacts with unique DNA response elements and activates transcription (23, 24). Although phenotypic analysis of Stat6-/- mice have elegantly demonstrated a role for Stat6 in IL-4-induced lymphocyte proliferation, Th2 helper T cell differentiation, immunoglobulin class switching, and cell surface antigen expression (27-29), the mechanism(s) by which Stat6 induces these effects remain incompletely understood.
The present invention provides two previously unknown human Stat6 variants, Stat6b and Stat6c, that are a naturally occurring, attenuated variant and a naturally occurring dominant negative variant, respectively, of Stat6. Also provided is the entire genomic sequence of the human Stat6 gene, including chromosomal mapping and genetic linkage analysis.
The present invention provides an isolated nucleic acid encoding the polypeptide Stat6b, having an amino acid sequence of Stat6 wherein at least 110 amino acids are deleted at the amino terminus, as well as an isolated nucleic acid encoding the polypeptide Stat6b, having the nucleotide sequence of SEQ ID NO:1.
Further provided is an isolated polypeptide, Stat6b, having an amino acid sequence of Stat6 wherein at least 110 amino acids are deleted from the amino terminus, an isolated polypeptide, Stat6b, having an amino acid sequence of Stat6 wherein amino acids 39-86 are deleted at the amino terminus and an isolated polypeptide, Stat6b, having the amino acid sequence of SEQ ID NO:2.
In addition, the present invention provides an isolated nucleic acid encoding the polypeptide Stat6c, having an amino acid sequence of Stat6 wherein amino acids 537-564 are deleted and an isolated nucleic acid encoding the polypeptide Stat6c, having the nucleotide sequence of SEQ ID NO:3.
Also provided is an isolated polypeptide, Stat6c, having an amino acid sequence of Stat6 wherein amino acids 537-564 are deleted and an isolated polypeptide, Stat6c, having the amino acid sequence of SEQ ID NO:4.
A method of producing the polypeptide Stat6b or the polypeptide Stat6c is also provided, comprising culturing cells containing a vector comprising nucleic acid encoding Stat6b or nucleic acid encoding Stat6c under conditions whereby the polypeptide Stat6b or the polypeptide Stat6c is produced.
As used herein, xe2x80x9caxe2x80x9d can include multiples.
The present invention provides the discovery of previously unknown isoforms of the human Stat6 gene, produced by differential splicing of the Stat6 gene, which have been identified as having distinct modulating functions within cells in which they are expressed. Specifically, a family of proteins termed signal transducers and activators of transcription (Stat) is known. Among the members of the Stat family is the protein Stat6 which has been isolated and cloned (See, ref 39 or U.S. Pat. No. 5,591,825 (McKnight, et al., issued Jan. 7, 1997)). Stat6 has been shown to play a role in interleukin-4 (IL-4) mediated signaling and may play a role in lymphoid cell proliferation and transcription. In studying the role of Stat6, two previously unknown, naturally occurring isoforms of Stat6 of the present invention, Stat6b and Stat6c, have been isolated and cloned. Any reference below to particular codons or base pairs of human Stat6 in describing the sequence of Stat6b or Stat6c are derived from the publicly available cDNA sequence of Stat6 as provided in SEQ ID NO:1 of U.S. Pat. No. 5,591,825 issued Jan. 7, 1997 or from the genomic sequence of human Stat6 provided herein as SEQ ID NO:5 and the cDNA sequence of wild type human Stat6 provided herein as SEQ ID NO:67.
The present invention provides an isolated nucleic acid encoding the polypeptide Stat6b, having an amino acid sequence of Stat6 wherein at least 110 amino acids are deleted at the amino terminus. For example, the nucleic acid can have a deletion encompassing the last base pair of codon 39 of Stat6 and continuing through to and including codon 86 of Stat6. Additionally, the nucleic acid of this invention can be an isolated nucleic acid encoding the polypeptide Stat6b, having the nucleotide sequence of SEQ ID NO:1.
In addition, the present invention provides an isolated nucleic acid encoding the polypeptide Stat6c, having an amino acid sequence of Stat6 wherein amino acids 537-564 are deleted. For example, the nucleic acid encoding the polypeptide Stat6c can have a deletion encompassing the last base pair of codon 536 of Stat6 and continuing through to and including the first two base pairs of codon 564 of Stat6. In addition, the present invention provides an isolated nucleic acid encoding the polypeptide Stat6c, having the nucleotide sequence of SEQ ID NO:3.
xe2x80x9cNucleic acidxe2x80x9d as used herein refers to single- or double-stranded molecules which may be DNA, comprised of the nucleotide bases A, T, C and G, or RNA, comprised of the bases A, U (substitutes for T) , C, and G. The nucleic acid may represent a coding strand or its complement. Nucleic acids may be identical in sequence to the sequence which is naturally occurring or may include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence (39). Furthermore, nucleic acids may include codons which represent conservative substitutions of amino acids as described in Table I.
As used herein, the term xe2x80x9cisolatedxe2x80x9d means a nucleic acid separated or substantially free from at least some of the other components of the naturally occurring organism, for example, the cell structural components commonly found associated with nucleic acids in a cellular environment and/or other nucleic acids. The isolation of nucleic acids can therefore be accomplished by techniques such as cell lysis followed by phenol plus chloroform extraction, followed by ethanol precipitation of the nucleic acids (30). The nucleic acids of this invention can be isolated from cells according to methods well known in the art for isolating nucleic acids. Alternatively, the nucleic acids of the present invention can be synthesized according to standard protocols well described in the literature for synthesizing nucleic acids.
The nucleic acid of this invention can be used as a probe or primer to identify the presence of a nucleic acid encoding the Stat6b or Stat6c polypeptide in a sample.
The nucleic acid of this invention can also be used as a probe or primer to distinguish nucleic acid encoding Stat6 from nucleic acid encoding Stat6b and/or Stat6c. Thus, the present invention also provides a nucleic acid having sufficient complementarity to the Stat6b and/or Stat6c nucleic acid of this invention to selectively hybridize with the Stat6b and/or Stat6c nucleic acid of this invention under stringent conditions as described herein and which does not hybridize with Stat6 nucleic acid under stringent conditions.
xe2x80x9cStringent conditionsxe2x80x9d refers to the hybridization conditions used in a hybridization protocol or in the primer/template hybridization in a PCR reaction. In general, these conditions should be a combination of temperature and salt concentration for washing chosen so that the denaturation temperature is approximately 5-20xc2x0 C. below the calculated Tm (melting/denaturation temperature) of the hybrid under study. The temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference nucleic acid are hybridized to the primer nucleic acid of interest and then amplified under conditions of different stringencies. The stringency conditions are readily tested and the parameters altered are readily apparent to one skilled in the art. For example, MgCl2 concentrations used in PCR buffer can be altered to increase the specificity with which the primer binds to the template, but the concentration range of this compound used in hybridization reactions is narrow, and therefore, the proper stringency level is easily determined. For example, hybridizations with oligonucleotide probes 18 nucleotides in length can be done at 5-10xc2x0 C. below the estimated Tm in 6xc3x97SSPE, then washed at the same temperature in 2xc3x97SSPE (40). The Tm of such an oligonucleotide can be estimated by allowing 2xc2x0 C. for each A or T nucleotide, and 4xc2x0 C. for each G or C. An 18 nucleotide probe of 50% G+C would, therefore, have an approximate Tm of 54xc2x0 C. Likewise, the starting salt concentration of an 18 nucleotide primer or probe would be about 100-200 mM. Thus, stringent conditions for such an 18 nucleotide primer or probe would be a Tm of about 54xc2x0 C. and a starting salt concentration of about 150 mM and modified accordingly by preliminary experiments. Tm values can also be calculated for a variety of conditions utilizing commercially available computer software (e.g., OLIGO(copyright)).
Modifications to the nucleic acids of the invention are also contemplated as long as the essential structure and function of the polypeptide encoded by the nucleic acids is maintained. Likewise, fragments used as primers can have substitutions so long as enough complementary bases exist for selective amplification (44) and fragments used as probes can have substitutions so long as enough complementary bases exist for hybridization with the reference sequence to be distinguished from hybridization with other sequences.
Probes of this invention can be used, for example, to screen genomic or cDNA libraries or to identify complementary sequences by Northern and Southern blotting. Primers of this invention can be used, for example, to transcribe cDNA from RNA and to amplify DNA according to standard amplification protocols, such as PCR, which are well known in the art.
Thus, the present invention further provides a method of detecting the expression of Stat6 and/or a Stat6b and/or Stat6c isoform in cells in a biological sample by detecting mRNA for Stat6 and/or Stat6b and/or Stat6c in the cells comprising the steps of: contacting the cells with a detectably labeled nucleic acid probe that hybridizes, under stringent conditions, with mRNA for Stat6b and not with mRNA for Stat6 or Stat6c and/or contacting the cells with a detectably labeled nucleic acid probe that hybridizes, under stringent conditions, with mRNA for Stat6c and not with mRNA for Stat6 or Stat6c: and detecting the presence of mRNA and/or contacting the cells with a detectably labeled nucleic acid probe that hybridizes, under stringent conditions, with mRNA for Stat6 and not with mRNA for Stat6b or Stat6c. The mRNA of the cells in the biological sample can be contacted with the probe and detected according to protocols standard in the art for detecting mRNA, such as Northern blotting and PCR amplification. The detection and/or quantitation of DNA or mRNA encoding Stat6 and/or Stat6b and/or Stat6c can be used to detect differential expression of Stat6 isoforms in a wide variety of diseases, including, for example, but not limited to, myeloid cancer, asthma, sarcoma, scleroderma, bone marrow fibrosis, fibrotic diseases and acquired immune deficiency syndrome.
The nucleic acid encoding the polypeptide Stat6b or the polypeptide Stat6c of this invention as described herein can be part of a recombinant nucleic acid comprising any combination of restriction sites and/or functional elements as are well known in the art which facilitate molecular cloning and other recombinant DNA manipulations. Thus, the present invention further provides a recombinant nucleic acid comprising the nucleic acid encoding Stat6b or Stat6c of the present invention. In particular, the isolated nucleic acid encoding Stat6b or Stat6c and/or the recombinant nucleic acid comprising a nucleic acid encoding Stat6b can be present in a vector and vector can be present in a cell, which can be a cell cultured in vitro or a cell in a transgenic animal.
Thus, the present invention further provides a composition comprising a vector comprising a nucleic acid encoding Stat6b and a vector comprising a nucleic acid encoding Stat6c and a vector comprising nucleic acid encoding both Stat6b and Stat6c. The composition can be in a pharmaceutically acceptable carrier. The vector can be an expression vector which contains all of the genetic components required for expression of the nucleic acid encoding Stat6b and/or the nucleic acid encoding Stat6c in cells into which the vector has been introduced, as are well known in the art. The expression vector can be a commercial expression vector or it can be constructed in the laboratory according to standard molecular biology protocols. The expression vector can comprise viral nucleic acid including, but not limited to, adenovirus, retrovirus vaccinia virus and/or adeno-associated virus nucleic acid. The nucleic acid or vector of this invention can also be in a liposome or a delivery vehicle which can be taken up by a cell via receptor-mediated or other type of endocytosis.
The present invention further provides the entire genomic sequence of the human Stat6 gene as SEQ ID NO:5. The genomic sequence of the Stat6 gene can be used for producing probes for identifying introns and exons and intron/exon junctions by hybridization and for producing sequencing primers. In particular, the 5xe2x80x2 end of the genomic sequence contains the Stat6 promoter which can be employed in assays to determine the therapeutic value of cis/trans regulation of the promoter by various growth factors, cytokines, lymphokines and chemokines which can be analyzed for this type of regulatory effect on the Stat6 promoter. In addition, identification of the intron sequences which regulate Stat6 splicing could provide a unique approach to regulating the expression levels of Stat6 isoforms.
In addition, the present invention provides the genetic locus of the human Stat6 gene as chromosome 12q bands 13.3-14.1. The identification of the Stat6 locus thus provides for detection of chromosomal aberrations and translocations involving the Stat6 gene. In particular, the 12q13 amplicon is dubbed the tumor specific amplicon (12q13), due to the high numbers of chromosomal aberrations/translocations affecting this locus that have been reported in a wide variety of tumors. The localization of the Stat6 gene to this region indicates that translocations/amplification/break points affecting the Stat6 gene can be found in a wide variety of tumors as well.
The present invention also provides an isolated polypeptide, Stat6b, having an amino acid sequence of Stat6 wherein at least 110 amino acids are deleted from the amino terminus, an isolated polypeptide, Stat6b, having an amino acid sequence of Stat6 wherein amino acids 39-86 are deleted at the amino terminus and an isolated polypeptide, Stat6b, having the amino acid sequence of SEQ ID NO:2.
Further provided is an isolated polypeptide, Stat6c, having an amino acid sequence of Stat6 wherein amino acids 537-564 are deleted and an isolated polypeptide, Stat6c, having the amino acid sequence of SEQ ID NO:4.
The present invention further provides a method of producing the polypeptide Stat6b or the polypeptide Stat6c, comprising culturing the cells of the present invention which contain a nucleic acid encoding the polypeptide Stat6b or nucleic acid encoding the polypeptide Stat6c under conditions whereby the polypeptide Stat6b or the polypeptide Stat6c is produced. Conditions whereby the polypeptide Stat6b or Stat6c is produced can include the standard conditions of any expression system, either in vitro or in vivo, in which the polypeptides of this invention are produced in functional form. For example, protocols describing the conditions whereby nucleic acids encoding the Stat6b or Stat6c proteins of this invention are expressed are provided in the Examples section herein. The polypeptide Stat6b or Stat6c can be isolated and purified from the cells according to methods standard in the art.
Specifically a method of producing the polypeptide Stat6b is provided, comprising culturing cells comprising vectors comprising a nucleic acid selected from the group consisting of: a) an isolated nucleic acid encoding the polypeptide Stat6b, having an amino acid sequence of Stat6 wherein at least 110 amino acids are deleted at the amino terminus, b) an isolated nucleic acid encoding the polypeptide Stat6b, having an amino acid sequence of Stat6 wherein a deletion in the nucleic acid is present, encompassing the last base pair of codon 39 of Stat6 and continuing through codon 86 of Stat6, inclusive, and an isolated nucleic acid encoding the polypeptide Stat6b, having the nucleotide sequence of SEQ ID NO:1, under conditions whereby the polypeptide Stat6b is produced.
Also provided is a method of producing the polypeptide Stat6c, comprising culturing cells comprising vectors comprising a nucleic acid selected from the group consisting of: a) an isolated nucleic acid encoding the polypeptide Stat6c, having an amino acid sequence of Stat6 wherein amino acids 537-564 are deleted, b) an isolated nucleic acid encoding the polypeptide Stat6c, having an amino acid sequence of Stat6 wherein a deletion in the nucleic acid is present, encompassing the last base pair of codon 536 of Stat6 and continuing through the first two base pairs of codon 564 of Stat6, inclusive, and an isolated nucleic acid encoding the polypeptide Stat6c, having the nucleotide sequence of SEQ ID NO:3, under conditions whereby the polypeptide Stat6c is produced.
In addition, the present invention provides peptides present in the wild type Stat6 sequence which are not present in the isoforms Stat6b or Stat6c. Specifically, the first 110 amino acids at the amino terminus of the wild type Stat6 polypeptide and/or amino acids 39-85 of wild type Stat6, as shown in SEQ ID NO:68 herein can be used to distinguish the presence of Stat6 and Stat6b on the basis that these amino acid sequences, which are present in Stat6, are not present in Stat6b. Thus, the peptides representing the 110 amino acids at the amino terminus of the wild type Stat6 polypeptide and/or amino acids 39-85 of wild type Stat6 can be used to produce antibodies which specifically bind Stat6 but do not bind Stat6b. Additionally, the nucleic acid sequence, encoding peptides representing the 110 amino acids at the amino terminus of the wild type Stat6 polypeptide and/or encoding amino acids 39-85 of wild type Stat6, as shown in SEQ ID NO:67 herein, can be used as probes and/or primers to distinguish a nucleic acid encoding a Stat6 polypeptide from a nucleic acid encoding a Stat6b polypeptide.
Similarly, amino acids 537-564 of the wild type Stat6 polypeptide as shown in SEQ ID NO:68 herein can be used to distinguish the presence of Stat6 and Stat6c on the basis that these amino acid sequences, which are present in Stat6, are not present in Stat6c. Thus, a peptide having amino acids 537-564 of wild type Stat6 can be used to produce antibodies which specifically bind Stat6 but do not bind Stat6c. Additionally, the nucleic acid sequence, encoding peptides representing amino acids 537-564 of wild type Stat6, as shown in SEQ ID NO:67 herein, can be used as probes and/or primers to distinguish a nucleic acid encoding a Stat6 polypeptide from a nucleic acid encoding a Stat6c polypeptide.
As used herein, xe2x80x9cisolatedxe2x80x9d and/or xe2x80x9cpurifiedxe2x80x9d means a polypeptide which is substantially free from the naturally occurring materials with which the polypeptide is normally associated in nature. Also as used herein, xe2x80x9cpolypeptidexe2x80x9d refers to a molecule comprised of amino acids which correspond to those encoded by a nucleic acid. The polypeptides of this invention can consist of the entire amino acid sequence of the Stat6b or Stat6c protein or portions thereof that are distinguishable from portions of the wild type Stat6 polypeptide. The polypeptides or portions thereof of the present invention can be obtained by isolation and purification of the polypeptides from cells where they are produced naturally or by expression of exogenous DNA encoding the Stat6b or Stat6c polypeptide. Portions of the Stat6b or Stat6c polypeptides can be obtained by chemical synthesis of peptides, by proteolytic cleavage of the polypeptides and by synthesis from nucleic acid encoding the portion of interest The polypeptide may include conservative substitutions where a naturally occurring amino acid is replaced by one having similar properties. Such conservative substitutions do not alter the function of the polypeptide and would be understood to include at least those listed in Table 1. (41).
Thus, it is understood that, where desired, modifications and changes may be made in the nucleic acid and/or amino acid sequence of the Stat6b and Stat6c polypeptides of the present invention and still obtain a protein having like or otherwise desirable characteristics. Such changes may occur in natural isolates or may be synthetically introduced using site-specific mutagenesis, the procedures for which, such as mis-match polymerase chain reaction (PCR), are well known in the art.
For example, certain amino acids may be substituted for other amino acids in a Stat6b or Stat6c polypeptide without appreciable loss of functional activity. Since it is the interactive capacity and nature of a protein that defines that protein""s biological functional activity, certain amino acid sequence substitutions can be made in a Stat6b or Stat6c amino acid sequence (or, of course, the underlying nucleic acid sequence) and nevertheless obtain a Stat6b or Stat6c polypeptide with like properties. It is thus contemplated that various changes may be made in the amino acid sequence of the Stat6b or Stat6c polypeptide (or underlying nucleic acid sequence) without appreciable loss of biological utility or activity and possibly with an increase in such utility or activity.
The present invention further provides antibodies which specifically bind the Stat6 isoforms Stat6b or Stat6c and do not bind Stat6. For example, antibodies which specifically bind Stat6c but do not bind Stat6b or Stat6 are made according to standard protocols from a peptide synthesized by standard protocols having the amino acid sequence LRSYWSDRDSEIGGIT (SEQ ID NO:66). In addition, antibodies which specifically bind Stat6 but do not bind Stat6b or Stat6c, as well as antibodies which specifically bind Stat6b but do not bind Stat6 or Stat6c, can be produced by well known methods for polyclonal and monoclonal antibody production according to the teachings provided herein.
The antibodies of the present invention include both polyclonal and monoclonal antibodies. Such antibodies may be murine, fully human, chimeric or humanized. These antibodies also include Fab or F(abxe2x80x2)2 fragments. The antibodies can be of any isotype IgG, IgA, IgD, IgE and IgM. Such antibodies can be produced by techniques well known in the art which include those described in Kohler et al. (42) or U.S. Pat. Nos. 5,545,806, 5,569,825 and 5,625,126, incorporated herein by reference.
The antibodies of this invention can be used to detect the presence of Stat6 and/or Stat6b and/or Stat6c in a sample. For example, a method is provided for detecting a Stat6b protein or antigen in a sample, which can be a biological sample, comprising contacting the sample with an antibody which specifically binds Stat6b and which does not bind Stat6c or Stat6, under conditions whereby an antigen/antibody complex can form and detecting the presence of the complex, whereby the presence of the antigen/antibody complex indicates the presence of a Stat6b protein or antigen in the sample.
In addition, a method is provided for detecting a Stat6c protein or antigen in a sample, which can be a biological sample, comprising contacting the sample with an antibody which specifically binds Stat6c and which does not bind Stat6b or Stat6, under conditions whereby an antigen/antibody complex can form and detecting the presence of the complex, whereby the presence of the antigen/antibody complex indicates the presence of a Stat6c protein or antigen in the sample.
Furthermore, a method is provided for detecting a Stat6 protein or antigen in a sample, which can be a biological sample, comprising contacting the sample with an antibody which specifically binds Stat6 and which does not bind Stat6b or Stat6c, under conditions whereby an antigen/antibody complex can form and detecting the presence of the complex, whereby the presence of the antigen/antibody complex indicates the presence of a Stat6 protein or antigen in the sample.
The conditions whereby an antigen/antibody complex can form and be detected can be standard conditions well known in the art for protocols such as immunoprecipitation, agglutination, Western blotting etc. Examples of protocols for producing and detecting antigen/antibody complexes are provided in the Examples section herein.
The present invention further contemplates a method for detecting the presence of Stat6 or Stat6b or Stat6c polypeptide in a biological sample comprising: contacting a biological sample with an antibody which specifically binds Stat6, Stat6b and Stat6c under conditions whereby an antibody/protein complex can form; isolating the protein in the antibody/protein complex; contacting a first portion of the isolated protein with an antibody which binds Stat6 and Stat6c but does not bind Stat6b under conditions whereby a protein/antibody complex can form and detecting the presence of complex formation; and contacting a second portion of the isolated protein with an antibody which binds Stat6 and Stat6b but does not bind Stat6c under conditions whereby a protein/antibody complex can form and detecting the presence of complex formation, whereby the absence of complex formation with the first portion of the isolated protein and the presence of complex formation with the second portion indicates the presence of Stat6b in the sample and the presence of complex formation with the first portion of the isolated protein and the absence of complex formation with the second portion of the isolated protein indicates the presence of Stat6c in the sample and the presence of complex formation in both the first and second portions indicates the presence of Stat6 in the sample.
For example, to produce an antibody which specifically binds Stat6 and Stat6b, but does not bind Stat6c, a peptide can be synthesized which consists of some or all of the 28 amino acids which are present in Stat6 and Stat6b but are deleted from Stat6c, such as, for example, LIIGFISKQYVTSLLLNEPDGTFLLRFS (SEQ ID NO: 62) or FISKQYVTSLLLNEPDGT (SEQ ID NO:63). Such peptides can then be used to generate polyclonal or monoclonal antibodies according to standard protocols. To produce an antibody that specifically binds Stat6 and Stat6c but does not bind Stat6b, a peptide can be synthesized which consists of some or all of the amino acids which are present in Stat6 and Stat6c but are deleted from Stat6b, which are the first 110 amino acids at the amino terminus of Stat6. For example, peptides having the sequence MPPEKVQRLYVDFPQH (SEQ ID NO:64) or SDTVQHLQASVGEQGEGST (SEQ ID NO:65) can be used to generate polyclonal or monoclonal antibodies according to standard protocols.
As demonstrated by the data provided herein, Stat6b, when compared to Stat6, is an attenuated regulator of gene transcription. Stat6c is a dominant negative regulator of gene transcription. Due to the role of these variants in regulating gene transcription, the isolated and purified nucleic acids or amino acid sequences which encode Stat6b and Stat6c of the present invention can be used to study gene regulation and in screening assays for identifying drug candidates which may be agonists or antagonists of Stat6b and or Stat6c or of other molecular targets in the signaling pathways in which these molecules are involved.
Thus, the present invention additionally provides a bioassay for identifying agonists or antagonists of activity mediated by Stat6b and Stat6c, comprising contacting cells which express Stat6b or Stat6c with a substance to be identified as an agonist or antagonist of Stat6 isoform activity (e.g., growth factors, cytokines, chemokines, etc.) and assaying the cells for induction of Stat6b or Stat6c by various assays, such as, for example, RNase protection assay, RT-PCR amplification or immunodetection as are described herein and in the literature.
The present invention also contemplates the use of the Stat6b and Stat6c polypeptides of this invention in gene therapy protocols. In particular, Stat6b and/or Stat6c can therapeutically modulate the development and differentiation of B and T cells and can enhance IL-4 immunological function in immunocompromised individuals through B cell, T cell, mast cell and/or macrophage gene therapy. For example, Stat6b and/or Stat6c gene therapy can enhance the growth of CD4+ T lymphocytes killed during human immunodeficiency virus (HIV) infection. Likewise, targeted Stat6c dominant negative expression can reduce proliferation and inflammation associated with IL-4 functional response. Reduction of IL-4 responsiveness can help increase interferon-related immune responses. Furthermore, modulation of Stat6 activity can control the proliferation and differentiation of cell types involved in fibrotic disease such as arthritis, scleroderma, bone marrow fibrosis and lung fibrosis and in inflammatory responses associated with asthma. In addition, Stat6 has been demonstrated to be activated by PDGF which has been implicated in arthritis, atherosclerosis, fibrotic diseases and neoplasia. Stat6 agonists and/or antagonists can be screened as described herein for therapeutic potential for these molecules in controlling Stat6 function in these diseases and inflammatory responses, as well as in neoplasia and arteriosclerosis. For example, Stat6c gene therapy would be useful in treating any disease where Stat6 is involved in the disease process as well as to inhibit undesirable proliferative, inflammatory and differentiation effects mediated by Stat6.
Thus, the present invention also provides a method for delivering Stat6b and/or Stat6c to a cell comprising administering to the cell a nucleic acid encoding Stat6b and/or Stat6c under conditions whereby the nucleic acid is expressed, thereby delivering Stat6b and/or Stat6c to the cell. The nucleic acid can be delivered as naked DNA or in a vector (which can be a viral vector) or other delivery vehicles and can be delivered to the subject""s cells by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, viral infection, liposome fusion, endocytosis and the like). The cell can be any cell which can take up and express exogenous DNA and in particular can include, but is not limited to a lung tissue cell, myeloid cell, epithelial cell, B cell, T cell, mammary gland cell, mast cell, pancreas cell, kidney cell prostate cell and ovary cell.
The nucleic acid encoding Stat6b or Stat6c can be administered to the cells of the subject either in vivo and/or ex vivo. If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The nucleic acids of this invention can be introduced into the cells via any gene transfer mechanism, such as, for example, virus-mediated gene delivery, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.
The nucleic acid or vector of the present invention can also be administered in vivo in a pharmaceutically acceptable carrier. By xe2x80x9cpharmaceutically acceptablexe2x80x9d is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
In the methods described above which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), the nucleic acids of the present invention can be in the form of naked DNA or the nucleic acids can be in a vector for delivering the nucleic acids to the cells for expression of the Stat6b and/or Stat6c protein. The vector can be a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as well as other liposomes developed according to procedures standard in the art. In addition, the nucleic acid or vector of this invention can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.).
As one example, vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome (see e.g.,50,51). The recombinant retrovirus can then be used to infect and thereby deliver to the infected cells nucleic acid encoding Stat6b and/or Stat6c. The exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors (45), adeno-associated viral (AAV) vectors (46), lentiviral vectors (47) vaccinia viral vectors (57) and pseudotyped retroviral vectors (48). Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, 49). This invention can be used in conjunction with any of these or other commonly used gene transfer methods.
The nucleic acid or vector may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, although intravenous administration is typically preferred. The exact amount of the nucleic acid or vector required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disease being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every nucleic acid or vector. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein (see, e.g., Remington""s Pharmaceutical Sciences; ref 52).
Parenteral administration of the nucleic acid or vector of the present invention, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Pat. No. 3,610,795, which is incorporated by reference herein.
As one example, if the nucleic acid of this invention is delivered to the cells of a subject in an adenovirus vector, the dosage for administration of adenovirus to humans can range from about 107 to 109 plaque forming unit (pfu) per injection but can be as high as 1012 pfu per injection (53,54). Ideally, a subject will receive a single injection. If additional injections are necessary, they can be repeated at six month intervals for an indefinite period and/or until the efficacy of the treatment has been established.
In the methods of the present invention for the treatment of, for example, a fibrotic disorder, the efficacy of the treatment can be monitored according to clinical protocols well known in the art for monitoring the treatment of fibrotic disorders. For example, such clinical parameters as histopathological examination, which can entail immunohistochemical analysis for various markers such as extracellular matrix production, viementin, collagen and the presence of mesenchymal cells, can be monitored according to methods standard in the art. Ideally, these parameters would be measured at about ten days after gene delivery.
The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.